Chaperonin TRiC promotes the assembly of polyQ expansion proteins into nontoxic oligomers.

Aberrant folding and fibrillar aggregation by polyglutamine (polyQ) expansion proteins are associated with cytotoxicity in Huntington's disease and other neurodegenerative disorders. Hsp70 chaperones have an inhibitory effect on fibril formation and can alleviate polyQ cytotoxicity. Here we show that the cytosolic chaperonin, TRiC, functions synergistically with Hsp70 in this process and is limiting in suppressing polyQ toxicity in a yeast model. In vitro reconstitution experiments revealed that TRiC, in cooperation with the Hsp70 system, promotes the assembly of polyQ-expanded fragments of huntingtin (Htt) into soluble oligomers of approximately 500 kDa. Similar oligomers were observed in yeast cells upon TRiC overexpression and were found to be benign, in contrast to conformationally distinct Htt oligomers of approximately 200 kDa, which accumulated at normal TRiC levels and correlated with inhibition of cell growth. We suggest that TRiC cooperates with the Hsp70 system as a key component in the cellular defense against amyloid-like protein misfolding.

Proteolytic cleavage of polyglutamine-expanded ataxin-3 is critical for aggregation and sequestration of non-expanded ataxin-3.

Spinocerebellar ataxia type 3 (SCA3), like other polyglutamine (polyQ) diseases, is characterized by the formation of intraneuronal inclusions, but the mechanism underlying their formation is poorly understood. Here, we tested the "toxic fragment hypothesis", which predicts that proteolytic production of polyQ-containing fragments from the full-length disease protein initiates the aggregation process associated with inclusion formation and cellular dysfunction. We demonstrate that the removal of the N-terminus of polyQ-expanded ataxin-3 (AT3) is required for aggregation in vitro and in vivo. Consistently, proteolytic cleavage of full-length, pathogenic AT3 initiates the formation of sodium dodecylsulfate-resistant aggregates in neuroblastoma cells. Although full-length AT3 does not readily aggregate on its own, it is susceptible to co-aggregation with polyQ-expanded AT3 fragments. Interestingly, interaction with soluble polyQ-elongated fragments causes a structural distortion of wild-type AT3 prior to the formation of stable co-aggregates. These results establish the critical role of C-terminal, proteolytic fragments of AT3 in the molecular pathomechanism of SCA3, in strong support of the toxic fragment hypothesis.

The highly abundant protein Ag-lbp55 from Ascaridia galli represents a novel type of lipid-binding proteins.

Lipid-binding proteins exhibit important functions in lipid transport, cellular signaling, gene transcription, and cytoprotection. Their functional analogues in nematodes are nematode polyprotein allergens/antigens and fatty acid and retinoid-binding proteins. This work describes a novel 55-kDa protein, Ag-lbp55, purified from the parasitic nematode Ascaridia galli. By direct N-terminal sequencing, a partial amino acid sequence was obtained that allowed the design of oligonucleotide primers to obtain the full-length cDNA sequence. Sequence analysis revealed the presence of an N-terminal signal peptide of 25 amino acid residues and a FAR domain at the C terminus. Data base searches showed almost no significant homologies to other described proteins. The secondary structure of Ag-lbp55 was predominantly alpha-helical (65%) as shown by CD spectroscopy. It was found to bind with high affinity fatty acids (caprylic, oleic, and palmitic acid) and their fluorescent analogue dansylaminoundecanic acid. Immunolocalization showed that Ag-lbp55 is a highly abundant protein, mainly distributed in the inner hypodermis and extracellularly in the pseudocoelomatic fluid. A similar staining pattern was observed in other pathogenic nematodes, indicating the existence of similar proteins in these species.

PA-I lectin from Pseudomonas aeruginosa binds acyl homoserine lactones.

The study analyses the binding affinities of Pseudomonas aeruginosa PA-I lectin (PA-IL) to three N-acyl homoserine lactones (AHSL), quorum sensing signal molecules responsible for cell-cell communication in bacteria. It shows that like some plant lectins, PA-IL has a dual function and, besides its carbohydrate-binding capacity, can accommodate AHLS. Formation of complexes between PA-IL and AHSL with acyl side chains composed of 4, 6 or 12 methyl groups is characterized by changes in the emissions of two incorporated fluorescent markers, TNS and IAEDANS, both derivatives of naphthalene sulfonic acid. PA-IL shows increasing affinities to lactones with longer aliphatic side chains. The values of the apparent dissociation constants (K(d)), which are similar to the previously determined K(d) for the adenine high affinity binding, and the similar effects of lactones and adenine on the TNS emission indicate one identical binding site for these ligands, which is suggested to represent the central cavity of the oligomeric molecule formed after the association of the four identical subunits of PA-IL. Intramolecular distances between the fluorescent markers and protein Trp residues are determined by fluorescence resonance energy transfer (FRET).

Conformational and functional analysis of the lipid binding protein Ag-NPA-1 from the parasitic nematode Ascaridia galli.

Ag-NPA-1 (AgFABP), a 15 kDa lipid binding protein (LBP) from Ascaridia galli, is a member of the nematode polyprotein allergen/antigen (NPA) family. Spectroscopic analysis shows that Ag-NPA-1 is a highly ordered, alpha-helical protein and that ligand binding slightly increases the ordered secondary structure content. The conserved, single Trp residue (Trp17) and three Tyr residues determine the fluorescence properties of Ag-NPA-1. Analysis of the efficiency of the energy transfer between these chromophores shows a high degree of Tyr-Trp dipole-dipole coupling. Binding of fatty acids and retinol was accompanied by enhancement of the Trp emission, which allowed calculation of the affinity constants of the binary complexes. The distance between the single Trp of Ag-NPA-1 and the fluorescent fatty acid analogue 11-[(5-dimethylaminonaphthalene-1- sulfonyl)amino]undecanoic acid (DAUDA) from the protein binding site is 1.41 nm as estimated by fluorescence resonance energy transfer. A chemical modification of the Cys residues of Ag-NPA-1 (Cys66 and Cys122) with the thiol reactive probes 5-({[(2-iodoacetyl)amino]ethyl}amino) naphthalene-1-sulfonic acid (IAEDANS) and N,N'-dimethyl-N-(iodoacetyl)-N'-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)ethylenediamine (IANBD), followed by MALDI-TOF analysis showed that only Cys66 was labeled. The observed similar affinities for fatty acids of the modified and native Ag-NPA-1 suggest that Cys66 is not a part of the protein binding pocket but is located close to it. Ag-NPA-1 is one of the most abundant proteins in A. galli and it is distributed extracellularly mainly as shown by immunohistology and immunogold electron microscopy. This suggests that Ag-NPA-1 plays an important role in the transport of fatty acids and retinoids.

Cellular toxicity of polyglutamine expansion proteins: mechanism of transcription factor deactivation.

The expression of polyglutamine-expanded mutant proteins in Huntington's disease and other neurodegenerative disorders is associated with the formation of intraneural inclusions. These aggregates could potentially cause cellular toxicity by sequestering essential proteins possessing normal polyQ repeats, including the transcription factors TBP and CBP. We show, in vitro and in cells, that monomers or small soluble oligomers of huntingtin exon1 accumulate in the nucleus and inhibit the function of TBP in a polyQ-dependent manner. FRET experiments indicate that these toxic forms are generated through a conformational rearrangement in huntingtin. Interaction of toxic huntingtin with the benign polyQ repeat of TBP structurally destabilizes the transcription factor, independent of the formation of insoluble coaggregates. Hsp70/Hsp40 chaperones interfere with the conformational change in mutant huntingtin and inhibit the deactivation of TBP. These results outline a molecular mechanism of cellular toxicity in polyQ disease and can explain the beneficial effects of molecular chaperones.

Fluorescence analysis of hormone binding activities of wheat germ agglutinin.

Wheat germ agglutinin (WGA) from embryos of the monocotyledonous plant Triticum vulgaris (Graminaceae) is a carbohydrate binding protein characterized by high specificity to N-acetyl-d-glucosamine and N-acetyl-d-neuraminic acid. In this study we show that parallel to its carbohydrate binding activities, WGA binds with several orders of magnitude higher affinity adenine, adenine-related cytokinins: kinetin, zeatin and isopentenyl-adenine as well as abscisic and gibberellic acids (K(d) 0.43-0.65 microM). Its interactions with these ligands cause conformational rearrangements in the protein molecules and significant enhancement of the protein tryptophan fluorescence (up to 60%) allowing characterization of the protein-hormone complexes. Dimeric WGA molecules possess two different classes of binding sites for the fluorescent hydrophobic probe 2-(p-toluidinyl) naphthalene sulfonic acid (TNS) as suggested by the sigmoid shape of the fluorescence titration curve and the value of the Hill coefficient (n(H) 1.6+/-0.3). The plant hormones displace part of the bound TNS probe and share the higher affinity TNS binding sites. These results characterize WGA as a hormone-binding protein.

Fluorescence analysis of the Hansenula polymorpha peroxisomal targeting signal-1 receptor, Pex5p.

Correct sorting of newly synthesized peroxisomal matrix proteins is dependent on a peroxisomal targeting signal (PTS). So far two PTSs are known. PTS1 consists of a tripeptide that is located at the extreme C terminus of matrix proteins and is specifically recognized by the PTS1-receptor Pex5p. We studied Hansenula polymorpha Pex5p (HpPex5p) using fluorescence spectroscopy. The intensity of Trp fluorescence of purified HpPex5p increased by 25% upon shifting the pH from pH 6.0 to pH 7.2. Together with the results of fluorescence quenching by acrylamide, these data suggest that the conformation of HpPex5p differs at these two pH values. Fluorescence anisotropy decay measurements revealed that the pH affected the oligomeric state of HpPex5p, possibly from monomers/dimers at pH 6.0 to larger oligomeric forms at pH 7.2. Addition of dansylated peptides containing a PTS1, caused some shortening of the average fluorescence lifetime of the Trp residues, which was most pronounced at pH 7.2. Our data are discussed in relation to a molecular model of HpPex5p based on the three-dimensional structure of human Pex5p.

Binding of hydrophobic ligands by Pseudomonas aeruginosa PA-I lectin.

The ability of Pseudomonas aeruginosa PA-I lectin to bind the fluorescent hydrophobic probe, 2-(p-toluidinyl) naphthalene sulfonic acid (TNS), and adenine was examined by spectrofluorametry and equilibrium dialysis. Interaction of TNS with PA-I caused significant enhancement of TNS fluorescence. The Hill coefficient (3.8+/-0.3) and the dissociation constant (8.7+/-0.16 microM) showed that TNS probably bound to four high affinity hydrophobic sites per PA-I tetramer. Interactions between PA-I and adenine were examined by equilibrium dialysis using [3H] adenine. The results indicated the presence of at least two classes of binding sites--one high and four lower affinity sites per tetramer with dissociation constants of 3.7+/-1.5 and 42.6+/-1.2 microM, respectively. These were distinct from the TNS sites as titration of TNS-equilibrated PA-I with adenine caused TNS fluorescence enhancement. The titration curve confirmed the existence of two classes of adenine-binding sites. Conversely, when PA-I was first equilibrated with adenine and then titrated with TNS, no TNS-binding was registered. This may indicate that conformational rearrangements of the lectin molecule caused by adenine prevent allosterically TNS binding.